Bacterial colonization in hospitalized patients is an important step in nosocomial infections. Frequent employment of antimicrobials can modify patients' normal microbiota, favoring colonization and infection by antimicrobial-resistant microorganisms. First-generation cephalosporins are frequently used as prophylactic antibiotics in surgery. Intestinal, oropharyngeal and skin colonization by cephalothin-resistant microorganisms were studied in 60 pre-operative patients at the Hospital Universitário Pedro Ernesto. Feces were cultured in Eosin-methylene blue medium containing 32 µg/mL of cephalothin. Swabs obtained from the oropharynx and from skin were inoculated in cistein-lactose electrolytes-deficient medium containing 32 µg/mL of cephalothin. Isolated strains were identified and tested for susceptibility to antimicrobials by disk diffusion. Cephalothin-resistant strains were isolated from the feces of 59 patients (98%), from the oropharynx of 13 patients (22%) and from skin in 10 patients (17%). Enterobacter cloacae was predominant in feces (68% of the patients) and oropharynx (13%). Acinetobacter spp. was the most frequent microorganism isolated from the skin (10%). Antimicrobial multiresistant strains were isolated from at least one of the sites in 38 patients (63%). The employment of selective medium containing antimicrobials is a relatively simple and efficient method, being useful to evaluate microorganisms from hospitalized patients' microbiota that are relevant as potential pathogens in nosocomial infections.